The invention concerns a method for the relative quantification of methylation of cytosine bases in DNA samples.
5-Methylcytosine is the most frequent covalently modified base in the DNA of eukaryotic cells. For example, it plays a role in the regulation of transcription, genomic imprinting and in tumorigenesis. The identification of 5-methylcytosine as a component of genetic information is thus of considerable interest. 5-Methylcytosine positions, however, cannot be identified by sequencing, since 5-methylcytosine has the same base pairing behavior as cytosine. In addition, in the case of a PCR amplification, the epigenetic information, which is borne by 5-methylcytosines, is completely lost.
Several methods are known for solving these problems. For the most part, a chemical reaction or enzymatic treatment of genomic DNA is performed, as a consequence of which cytosine can be distinguished from methylcytosine bases. A current method is the reaction of genomic DNA with bisulfite, which leads to a conversion of cytosine bases to uracil after alkaline hydrolysis in two steps (Shapiro, R., Cohen, B., Servis, R. Nature 227, 1047 (1970)). 5-Methylcytosine remains unchanged under these conditions. The conversion of C to U leads to a change in the base sequence, from which the original 5-methylcytosines can now be determined by sequencing (only methylcytosines can still provide a band in the C lane).
An overview of the other known possibilities for detecting 5-methylcytosines can be taken from the following review article together with all of the references cited therein: Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 26, 2255 (1998).
In the method described in DE 197 54 482 A1, knowledge of the frequency of methylation in a fragment is not directly obtained, but rather an abstract pattern is first obtained, the components of which need not necessarily belong to the initial fragments.
Paul, C. L. et al. (Biotechniques (1996) 21 (1) 126–33: Cytosine methylation: quantitation by automated genomic sequencing and GENESCAN analysis) describes a method of sequencing with the use of thymine and cytosine bases that are labeled differently. Presently, however, neither genomic sequencing nor an electrophoresis method, in general, are involved.
Yurov, Y. B. et al. (Human Genetics (1996) 97 (3) 390–8: High resolution multicolor fluorescence in situ hybridization using cyanine and fluorescein dyes: rapid chromosome identification by directly fluorescently labeled alphoid DNA probes) concerns the use of Cy3 and Cy5-dCTP for labeling purposes.
U.S. Pat. No. 5,837,832 describes arrays of oligonucleotides and their hybridization to sample DNA. However, either DNA methylation is not detected or the arrays described therein are not suitable for the hybridization of different fragments of complex amplifications so that they contain oligonucleotides complementary to the primers and thus specifically bind one fragment per oligonucleotide.
Different methods are known in the prior art, by means of which oligonucleotide arrays can be produced. They can be divided roughly into 3 groups:
1) All oligomers are prepared in the conventional manner individually and in relatively large quantities in special automated synthesis equipment and then individually pipetted onto the carrier. Automated, highly precise micropipette robot equipment is usually used for this purpose. The advantage of this method is that it is for the most part based on already optimized standard methods and equipment. Qualitatively superior DNA arrays with very pure oligomers can be produced in this way, which has an extremely positive influence on the detection sensitivity and reliability that can be obtained with the array. The great disadvantage of the method is that it is very time-consuming and is thus expensive.
2) The oligomers are synthesized by pipetting minimal quantities directly onto the substrate. The oligomer chain provided therein is constructed, nucleobase by nucleobase, at each grid point. For pipetting, as in method (1), a specialized micropipetting robot device is similarly used or, e.g., a device that contains channels for introducing the individual synthesis building blocks to the respective points of the array (EP-A-0915897). The chemical synthesis method is basically the same as for conventional oligomer synthesis in automated synthesis equipment.
3) The oligomers, as in method 2), are synthesized directly on the substrate, and the targeted binding of the correct nucleobases to the correct grid points is accomplished, however by a completely parallel photolithographic technique originating from semiconductor manufacture, instead of sequential, precisely targeted pipefting steps. The method is based on the fact that the 5′-OH protective groups can be removed from oligonucleotides in a targeted manner with light of a specific wavelength. By suitable local irradiation patterns, oligonucleotide ends can thus be made reactive at precisely those grid points at which it is desired that a new nucleotide building block will bind in the next step. When the array surface is completely wetted with a nucleotide building block solution, a nucleobase will thus be bound only at the previously exposed sites, and all of the unexposed sites will remain unchanged. The local exposure patterns are produced by positioning a photomicrograph black-and-white mask between the substrate and the light source that covers all grid sites, which will not be made reactive.
Due to the high parallel nature in processing, this method is very rapid and efficient, and it is also well suitable for the purpose of achieving very high grid densities, due to the high precision that can be obtained with photolithography.
An overview of the prior art relative to oligomer array production can also be taken from the special publication that appeared in January 1999 of Nature Genetics (Nature Genetics Supplement, Vol. 21, January 1999) and the literature cited therein.
Prior art, which generally concerns the use of oligomer arrays and photolithographic mask designs, includes, e.g., U.S. Pat No. 5,837,832; U.S. Pat No. 5,856,174, WO98/27430 and U.S. Pat No. 5,856,101.
The amplification by DNA by means of PCR is prior art.